ABSTRACT Neurite outgrowth measurements often require time-consuming measurements of multiple ima... more ABSTRACT Neurite outgrowth measurements often require time-consuming measurements of multiple images. The numerous experimental conditions lead to long analysis time and effort. A high-throughput assay provides a quick method to obtain quantitative measures of neurite outgrowth. In this chapter, the experimental setup and imaging protocol are described to efficiently process images of primary neurons or cell lines in order to quantitate parameters.
Proteoglycans in the central nervous system play integral roles as "traffic signals" fo... more Proteoglycans in the central nervous system play integral roles as "traffic signals" for the direction of neurite outgrowth. This attribute of proteoglycans is a major factor in regeneration of the injured central nervous system. In this review, the structures of proteoglycans and the evidence suggesting their involvement in the response following spinal cord injury are presented. The review further describes the methods routinely used to determine the effect proteoglycans have on neurite outgrowth. The effects of proteoglycans on neurite outgrowth are not completely understood as there is disagreement on what component of the molecule is interacting with growing neurites and this ambiguity is chronicled in an historical context. Finally, the most recent findings suggesting possible receptors, interactions, and sulfation patterns that may be important in eliciting the effect of proteoglycans on neurite outgrowth are discussed. A greater understanding of the proteoglycan-ne...
Glutamate transporters are responsible for removing glutamate from the extracellular space and ha... more Glutamate transporters are responsible for removing glutamate from the extracellular space and have the potential to protect neurons from excitotoxicity. In the present study, the effects of ceftriaxone and (2R, 4R)-APDC (APDC) on the protein expression of GLAST and GLT-1, the rate of glutamate uptake, and neuroprotection were evaluated in a cell culture model of glutamate excitotoxicity. Mixed neuron/astrocyte cultures were prepared from 1 day old rat pups. Protein levels of GLAST and GLT-1 glutamate transporters were quantified using In-Cell Western techniques after acute or 5-day treatment with either ceftriaxone or APDC. Glutamate uptake was measured using Michaelis-Menten kinetics to evaluate the effects of 5-day treatment with ceftriaxone or APDC. Neuronal cell death in response to a 10-minute 1 mM glutamate challenge was measured following 5-day treatment with either ceftriaxone or APDC. Five-day treatment with 100 μM ceftriaxone significantly increased both GLAST and GLT-1 p...
"Reactive" astr... more "Reactive" astrocytes and other glial cells in the injured CNS produce an altered extracellular matrix (ECM) that influences neuronal regeneration. We have profiled the glycosaminoglycan (GAG) component of proteoglycans (PGs) produced by reactive neonatal rat cortical astrocytes, and have quantified their neurite-outgrowth inhibitory activity. PGs extracted from cell layers and medium were fractionated on DEAE-Sephacel with a gradient of NaCl from 0.15 to 1.0 M. Monosaccharide analysis of the major peaks eluting at 0.6 M NaCl indicated an excess of GlcNH₂ to GalNH₂, suggesting an approximate HS/CS ratio of 6.2 in the cell layer and 4.2 in the medium. Chondroitinase ABC-generated disaccharide analysis of cell and medium PGs showed a >5-fold excess of chondroitin 4-sulfate over chondroitin 6-sulfate. Heparin lyase-generated disaccharides characteristic of the highly sulfated S-domain regions within HS were more abundant in cell layer than medium-derived PGs. Cell layer and medium HS disaccharides contained ~20% and ~40% N-unsubstituted glucosamine respectively, which is normally rare in HS isolated from most tissues. NGF-stimulated neurite outgrowth assays using NS-1 (PC12) neuronal cells on adsorbed substrata of PGs isolated from reactive astrocyte medium showed pronounced inhibition of neurite outgrowth, and aggregation of NS-1 cells. Cell layer PGs from DEAE-Sephacel pooled fractions having high charge density permitted greater NGF-stimulated outgrowth than PGs with lower charge density. Our results indicate the synthesis of both inhibitory and permissive PGs by activated astrocytes that may correlate with sulfation patterns and HS/CS ratios.
ABSTRACT Neurite outgrowth measurements often require time-consuming measurements of multiple ima... more ABSTRACT Neurite outgrowth measurements often require time-consuming measurements of multiple images. The numerous experimental conditions lead to long analysis time and effort. A high-throughput assay provides a quick method to obtain quantitative measures of neurite outgrowth. In this chapter, the experimental setup and imaging protocol are described to efficiently process images of primary neurons or cell lines in order to quantitate parameters.
Proteoglycans in the central nervous system play integral roles as "traffic signals" fo... more Proteoglycans in the central nervous system play integral roles as "traffic signals" for the direction of neurite outgrowth. This attribute of proteoglycans is a major factor in regeneration of the injured central nervous system. In this review, the structures of proteoglycans and the evidence suggesting their involvement in the response following spinal cord injury are presented. The review further describes the methods routinely used to determine the effect proteoglycans have on neurite outgrowth. The effects of proteoglycans on neurite outgrowth are not completely understood as there is disagreement on what component of the molecule is interacting with growing neurites and this ambiguity is chronicled in an historical context. Finally, the most recent findings suggesting possible receptors, interactions, and sulfation patterns that may be important in eliciting the effect of proteoglycans on neurite outgrowth are discussed. A greater understanding of the proteoglycan-ne...
Glutamate transporters are responsible for removing glutamate from the extracellular space and ha... more Glutamate transporters are responsible for removing glutamate from the extracellular space and have the potential to protect neurons from excitotoxicity. In the present study, the effects of ceftriaxone and (2R, 4R)-APDC (APDC) on the protein expression of GLAST and GLT-1, the rate of glutamate uptake, and neuroprotection were evaluated in a cell culture model of glutamate excitotoxicity. Mixed neuron/astrocyte cultures were prepared from 1 day old rat pups. Protein levels of GLAST and GLT-1 glutamate transporters were quantified using In-Cell Western techniques after acute or 5-day treatment with either ceftriaxone or APDC. Glutamate uptake was measured using Michaelis-Menten kinetics to evaluate the effects of 5-day treatment with ceftriaxone or APDC. Neuronal cell death in response to a 10-minute 1 mM glutamate challenge was measured following 5-day treatment with either ceftriaxone or APDC. Five-day treatment with 100 μM ceftriaxone significantly increased both GLAST and GLT-1 p...
"Reactive" astr... more "Reactive" astrocytes and other glial cells in the injured CNS produce an altered extracellular matrix (ECM) that influences neuronal regeneration. We have profiled the glycosaminoglycan (GAG) component of proteoglycans (PGs) produced by reactive neonatal rat cortical astrocytes, and have quantified their neurite-outgrowth inhibitory activity. PGs extracted from cell layers and medium were fractionated on DEAE-Sephacel with a gradient of NaCl from 0.15 to 1.0 M. Monosaccharide analysis of the major peaks eluting at 0.6 M NaCl indicated an excess of GlcNH₂ to GalNH₂, suggesting an approximate HS/CS ratio of 6.2 in the cell layer and 4.2 in the medium. Chondroitinase ABC-generated disaccharide analysis of cell and medium PGs showed a >5-fold excess of chondroitin 4-sulfate over chondroitin 6-sulfate. Heparin lyase-generated disaccharides characteristic of the highly sulfated S-domain regions within HS were more abundant in cell layer than medium-derived PGs. Cell layer and medium HS disaccharides contained ~20% and ~40% N-unsubstituted glucosamine respectively, which is normally rare in HS isolated from most tissues. NGF-stimulated neurite outgrowth assays using NS-1 (PC12) neuronal cells on adsorbed substrata of PGs isolated from reactive astrocyte medium showed pronounced inhibition of neurite outgrowth, and aggregation of NS-1 cells. Cell layer PGs from DEAE-Sephacel pooled fractions having high charge density permitted greater NGF-stimulated outgrowth than PGs with lower charge density. Our results indicate the synthesis of both inhibitory and permissive PGs by activated astrocytes that may correlate with sulfation patterns and HS/CS ratios.
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